Biomedical subjects
Publications and source records attributed to M Hutchings.
Activation of the tyrosine kinase ZAP 70 has been shown to be crucial to the transduction of the T-cell receptor signalling pathway, which leads ultimately to proliferation, cytokine gene expression and T-cell effector functions. A series of 2-phenylaminopyrimidines have been identified as potent and selective inhibitors of ZAP 70.
In this preclinical evaluation we have compared the efficacy of three clinical CD34+enrichment procedures with respect to purity, yield and recovery, as well as risk of selective loss of CD34+ lineage-specific subsets. The three devices work by different principles and have several different manipulation steps: The magnetic field separator uses paramagnetic iron-dextran particles; the magnetic microbead selection is based on the advantage of a large surface area for immobilisation of the monoclonal antibody within a very small volume; the original immunoabsorption technique is based on the use of biotinylated antibody applied to a column of avidin-coated sephadex beads. The results of this evaluation gave a median purity 96% (88-98%), 86% (62-97%), and 49% (18-85%), and median yield of 65% (54-100%), 40% (21-74%), and 30% (8-55%), respectively. Subset analysis recognised a selective loss of CD34+/61+ after enrichment, most likely due to class I-II antibodies used for the enrichment step or, alternatively, nonspecific binding of megakaryocytic progenitors. Tumour cell spiking experiments on a clinical scale documented an expected 2-4 log reduction resulting in a number of potentially malignant cells in the CD34 enriched product. Our data support four major conclusions: First, that magnetic field separation is superior to magnetic beads and chromatography selection, mainly due to the risk of cell loss and insufficient recovery with the two latter methods. Second, that late differentiated progenitors with CD34 class III epitopes present are lost during the enrichment procedures. The third major conclusion is that chromatography selection results in a selective loss of CD34bright cells, which are most likely uncommitted early progenitors. This was an unexpected finding which may be a consequence of an imbalance between the strong forces between biotin-avidin and insufficient physical manipulation for CD34+ cell release. Finally, the data document that CD34 selection alone is an inappropriate way to eliminate tumour cells due to the uncontrolled variables and the inconsistent outcome. The only products which can be expected to be purged free of tumour cells are the ones with very minimal (<10-5) contamination in the starting products, ie products documented tumour free with the most sensitive techniques for quantitation. If this is not the case, the optimal purging strategy may be a two-step procedure including CD34 selection and subsequent depletion of the tumour cells in question.
An obstacle to stem cell gene therapy for AIDS is the limited numbers of hematopoietic progenitors available. Granulocyte colony-stimulating factor (G-CSF) is used for mobilization of progenitors, but little is known about the functional characteristics of mobilized progenitors, and immature and T cell progenitors may not be mobilized. This study examined the effect of G-CSF on the function of progenitors. Ten human immunodeficiency virus-infected patients received G-CSF (filgrastim, 300 microgram/day) for 5 days. Absolute numbers of immature and T cell progenitors did not increase. The ability of CD34+ progenitor cells to generate lymphocytes was examined by use of thymic organ cultures. The mean number of lymphocytes generated per CD34+ cell on day 0 was 0.72 and on day 4 was 0.09 (P<.003). The number of CD4+ cells generated per CD34+ cell was significantly reduced after G-CSF treatment. Thus, G-CSF increased the number of mature progenitor cells but did not increase T cell progenitors.
Cerebral amyloid angiopathy in Alzheimer's disease is characterized by deposition of amyloid beta (Abeta) in cortical and leptomeningeal vessel walls. Although it has been suggested that Abeta is derived from vascular smooth muscle, deposition of Abeta is not seen in larger cerebral vessel walls nor in extracranial vessels. In the present study, we examine evidence for the hypothesis that Abeta is deposited in periarterial interstitial fluid drainage pathways of the brain in Alzheimer's disease and that this contributes significantly to cerebral amyloid angiopathy. There is firm evidence in animals for drainage of interstitial fluid from the brain to cervical lymph nodes along periarterial spaces; similar periarterial channels exist in humans. Biochemical study of 6 brains without Alzheimer's disease revealed a pool of soluble Abeta in the cortex. Histology and immunocytochemistry of 17 brains with Alzheimer's disease showed that Abeta accumulates five times more frequently around arteries than around veins, with selective involvement of smaller arteries. Initial deposits of Abeta occur at the periphery of arteries at the site of the putative interstitial fluid drainage pathways. These observations support the hypothesis that Abeta is deposited in periarterial interstitial fluid drainage pathways of the brain and contributes significantly to cerebral amyloid angiopathy in Alzheimer's disease.
Efficient gene transfer into hematopoietic stem cells offers a number of potential therapeutic applications. However, the relatively low titer of retroviral supernatants and the requirement for cell division to ensure integration have meant that transduction efficiency has been low. We have modified a flowthrough approach to cell transduction and have been able consistently to increase gene transfer efficiency into human hematopoietic progenitor cells. We transduced CD34 cells with retroviral vectors encoding a truncated nerve growth factor receptor (NGFR) or neo. Retroviral supernatant was pulled through 0.2-micron polycarbonated membranes, followed by placement of cells on the filter. In the absence of cytokines, the transduction efficiency of CD34 cells with a NGFR vector was increased 3-11-fold over that obtained at an identical MOI in liquid culture to produce 11%-44% transduction. Furthermore, both Thy1+ and Thy1- subsets in a total CD34 population were transduced with similar efficiency, and transduction with a neo vector, as measured by G418 resistance in clonogenic assays, increased 1.5-5-fold. The mechanism by which gene transfer is improved may reflect colocalization of cells and retrovirus. Costaining of cells transduced on the filter with an NGFR retrovirus with both an NGFR antibody and a gp70 antibody that recognizes viral coat protein revealed high-level coexpression. The levels of in vitro gene transfer we obtain are equivalent to those observed when CD34 cells are cocultured in liquid culture with cytokines. However, culture with cytokines may commit CD34 cells to differentiation and has produced disappointingly low levels of subsequent in vivo gene transfer. Gene marking studies using distinguishable retroviral vectors will provide a means of learning whether the effects of flowthrough transduction genuinely enhance the efficiency of gene transfer to human marrow-repopulating cells.
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There is evidence for lymphatic drainage of interstitial fluid from the brain along perivascular spaces in a number of mammalian species. Ultrastructural studies suggest that there are similar drainage pathways in the human cerebral cortex. Perivascular spaces in the basal ganglia, however, differ from those in the cortex in that they dilate to form lacunes and rarely accumulate beta-amyloid (amyloid angiopathy) in Alzheimer's disease; in the cortex, lacunes are rare but amyloid angiopathy is common. The aim of the present study is to compare the structure of perivascular spaces in the basal ganglia and at the anterior perforated substance with perivascular spaces in the cerebral cortex. Eight postmortem brains from patients aged 23-80 years (mean 68 y) were examined by light microscopy, by scanning and transmission electron microscopy and by direct visualisation of etched paraffin blocks. The results show that arteries in the basal ganglia are surrounded by 2 distinct coats of leptomeninges separated by a perivascular space which is continuous with the perivascular space around arteries in the subarachnoid space. The inner layer of leptomeninges closely invests the adventitia of the vessel wall and the outer layer is continuous with the pia mater on the surface of the brain at the anterior perforated substance. Veins in the basal ganglia have no outer layer of leptomeninges and thus the perivascular space is continuous with the subpial space. The anatomy of the periarterial spaces in the basal ganglia differs significantly from that in the cerebral cortex where there is only a single periarterial layer of leptomeninges. Differences in structure of perivascular spaces around arteries may reflect relative efficiencies in the drainage of interstitial fluid from different sites in the brain. Furthermore, the structure of the perivascular spaces may contribute to the relatively high frequency of lacunes in the basal ganglia, and the low frequency of amyloid angiopathy at this site in Alzheimer's disease.
We here present the case of a 70-year-old woman referred to our unit for investigation of bleeding. Investigations confirmed a high titre acquired Factor VIII inhibitor. In association there was relapse of systemic illness associated with anti-neutrophil cytoplasmic antibodies (atypical pattern) for which she had been treated five years previously. Immunosuppression was attempted, but it failed to have an impact both on the inhibitor titre and on the underlying disorder. The patient died from multi-organ failure and massive chest hemorrhage. Post-mortem showed necrotizing vasculitis of medium sized vessels at several sites, including the kidney, consistent with a diagnosis of polyarteritis nodosa. Although it is well recognised that Factor VIII inhibitors are found in conjunction with autoimmune disorders, this case is significant in that it is the first associated with histologically proven polyarteritis nodosa type vasculitis. The case illustrates the difficulties in the investigation and management of patients with acquired high titre Factor VIII inhibitors.
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OBJECTIVE: The purpose of this study was to correlate normal brain anatomy as seen on posterior fontanelle cranial sonography with anatomical sections of the premature infant brain. MATERIALS AND METHODS: Images obtained from 93 cranial ultrasound examinations performed via both the anterior and posterior fontanelle in 53 infants, ranging in gestational age from 24 to 42 weeks, were reviewed to determine the ultrasound anatomy visible and also the changing appearances with increasing gestational age. The brains of five infants were sectioned at post-mortem according to predetermined anatomical landmarks to correlate with posterior fontanelle ultrasound scan planes. Brain preservation techniques involved fixation in formalin at room temperature, refrigeration of brain following formalin fixation, and brain freezing at -17 degrees C. RESULTS: In the premature infant brain, the subarachnoid space is up to 15 mm in thickness. Occipital lobe anatomy well seen includes occipital horns of lateral ventricles, and white matter tracts to the visual cortex and visual association areas. Brain anatomy was better appreciated on sections obtained following brain freezing rather than formalin fixation. CONCLUSION: Satisfactory ultrasound anatomic correlation of the premature brain is possible using a brain freezing preservation technique. Posterior fontanelle ultrasound allows detailed illustration of occipital lobe anatomy.
In vitro and in vivo results obtained from a novel flexible amperometric oxygen sensor are reported. The sensor is fabricated using thin film deposition techniques and is operated by the application of a pulsed waveform. Development of the sensor was undertaken in order to produce a device that is capable of being sited at the interface of a wound and an overlying wound dressing. Oxygen determinations in such an environment would aid in gaining an understanding of the role of oxygen in wound healing and the type of wound dressing that would provide an environment conducive towards wound healing. In vitro data indicate that linearity of response is good although other performance characteristics are irreproducible. In vivo response to oxygen has been observed 50 h after insertion into a porcine sham wound. Expected trends were followed when changes to the oxygen regime of the wound space were effected, but absolute values of oxygen tension are difficult to state with certainty. This may be due to poor calibration stability and inadequate sealing of the sensor from the surrounding environment.
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This study of bilharzia and other intestinal parasites was carried out on 496 children in the Firaisana of Ankilivalo. As well as determine the prevalence of these parasites, data was collected on nutrition, agriculture and the use of water in order to gain an understanding of the transmission and effects of these parasites, and hence make recommendations for their control. In two schools within the area of a major irrigation scheme the prevalence of bilharzia was 69%, and 50% of children suffer from at least one other intestinal worm. In a school outside the main irrigation area, the prevalence was much lower (7%), but nutritional standards were also lower. Ultimately, control of bilharzia will depend on improvements to the irrigation and drainage infrastructure, and in standards of sanitation. However, chemotherapy is the only method of bringing the disease under control in the short term.
Using scanning and transmission electron microscopy and light microscopy, the authors studied the human pia mater and its relationship to the entry of blood vessels into the normal cerebral cortex. The purpose of this investigation was to examine the long-established concept that the subarachnoid space communicates directly with the perivascular spaces of the cerebral cortex. Brains obtained post mortem from subjects with recent subarachnoid hemorrhage (SAH) and purulent leptomeningitis were studied by light microscopy to determine the permeability of the pia mater to red blood cells and inflammatory cells. Scanning electron microscopy showed that the normal pia mater is a flat sheet of cells that is reflected from the surface of the brain to form the outer coating of the meningeal vessels in the subarachnoid space. Transmission electron microscopy confirmed that the cells of the pia mater are joined by junctional complexes and form a continuous sheet that separates the subarachnoid space on one side from the subpial and perivascular spaces on the other. Thus, neither the pia mater nor the subarachnoid space extends into the brain beside blood vessels as they enter the cerebral cortex. The perivascular spaces were, in fact, found to be confluent with the subpial space and not with the subarachnoid space. In cases of recent SAH, red blood cells did not enter the perivascular spaces from the subarachnoid space; neither did India ink injected post mortem into the subarachnoid space pass into the perivascular spaces. The results of these crude tracer studies suggest that the pia mater is an effective barrier to the passage of particulate matter. Histological examination of brains of patients who had died with purulent leptomeningitis showed that inflammatory cells were present in the cortical perivascular spaces and in the contiguous subpial spaces. The presence of a large number of inflammatory cells in the subarachnoid space suggests that inflammatory cells readily penetrate the pia mater that separates the perivascular spaces from the subarachnoid space. The permeability of the pia mater to small molecular weight substances is briefly discussed.